Nucleotide sequences of HIV-1 group (or subgroup) O retroviral antigens

ABSTRACT

An HIV-1 type (or subtype) O retrovirus protein, or a natural or synthetic polypeptide or peptide including at least a part of said protein, which is capable of being recognized by antibodies isolated from a serum resulting from infection by an HIV-1 type O VAU strain or an HIV-1 type (or subtype) O DUR strain.

This is a division of application Ser. No. 10/026,741, filed Dec. 27,2001 now U.S. Pat. No. 7,157,225, which is a Continuation 08/817,441,filed Jul. 11, 1997 now U.S. Pat. No. 6,399,294, which is the NationalPhase Application filed under 35 U.S.C. §371 of InternationalApplication No. PCT/FR95/01391, filed Oct. 20, 1995, all of which areincorporated herein by reference.

The invention relates to the antigens obtained by expression ofnucleotide sequences or by chemical synthesis, for example using AppliedBiosystems brand synthesizers, present in HIV-1 group (or subgroup) Ovariants and more particularly the antigens corresponding to those whichmay be isolated from viral particles. By way of example of HIV-1 virusesof the subgroup O, reference is made to the HIV-1_((VAU)) isolate and tothe HIV-1_((DUR)) isolate.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. The ASCII copy, created on May 26, 2010, is named 34953752.txtand is 77,322 bytes in size.

The invention also relates to monoclonal or polyclonal antibodiesinduced by these antigens.

The invention also relates to cloned DNA sequences either havingsequence analogy or complementarity with the genomic RNA of theabovementioned virus. The invention also relates to processes for thepreparation of these cloned DNA sequences. The invention also relates topolypeptides containing amino acid sequences coded for by the cloned DNAsequences.

Furthermore, the invention relates to applications of the antigensmentioned above to the in vitro detection in at-risk individuals ofcertain forms of AIDS and, as regards some of them, to the production ofimmunogenic compositions and vaccinating compositions against thisretrovirus. Similarly, the invention relates to applications of theabovementioned antibodies for the same purposes and, for some of them,to their application to the production of active principles formedicinal products against this human AIDS.

The invention also relates to the application of the cloned DNAsequences and of the polypeptides obtained from these sequences asprobes or primers for gene amplification, in diagnostic kits.

The invention also relates to antigenic compositions which may beobtained by chemical synthesis or by expression in a recombinant cellhost and which allow the diagnosis of an infection due to a humanretrovirus of HIV type independently of the HIV-1 or HIV-2 subtype. Suchcompositions comprise at least one peptide chosen from the antigenicpeptides common to the HIV-1, HIV-2, HIV-1_((DUR)) and HIV-1_((VAU))viruses or variants of the antigenic peptides possessing similarimmunogenic characteristics.

The invention is also directed toward compositions which allow thespecific diagnosis of an infection due to a human retrovirus of HIV-1type, more particularly HIV-1, group M, HIV-2 or HIV-1 group (orsubgroup) O and comprising at least one antigenic peptide specific forthe HIV-1 virus, an antigenic peptide specific for the HIV-2 virus andan antigenic peptide specific for the HIV-1 group (or subgroup) O virusor variants of these antigenic peptides possessing similar immunogeniccharacteristics. More particularly, the antigenic peptides are derivedfrom the envelope protein of HIV-1 group M and HIV-2 and HIV-1 group (orsubgroup) O viruses.

The invention is moreover directed toward a peptide allowing detectionof anti-HIV antibodies which the peptides of the prior art did notalways make it possible to detect, based in particular on the discoveryof a new HIV-1 strain: HIV-1 DUR. The antiserum directed against it doesnot always have reactivity with the peptides of the consensus HIV as itis used nowadays. The term “consensus HIV” refers to the regions whichare conserved between isolates and whose demonstration is essential tothe design of diagnostic reagents or vaccines, and whose mutationsimpart resistance to antiviral medicinal products. The term “peptide”used in the present text defines both oligopeptides and polypeptides.

STATE OF THE ART

Two types of human immunodeficiency virus (HIV) which haveresponsibility for the development of an LAS or AIDS have been isolatedand characterized. A first virus, known as LAV-1 or HIV-1, was isolatedand described in GB patent application 8324,800 and patent applicationEP 84401,834 of 14 Sep. 1984. This virus was also described by F.Barré-Sinoussi et al. in Science (1983), 220, 868-871.

The type 2 HIV retrovirus belongs to a separate class and has only alimited immunological relationship with type 1 HIV retroviruses. HIV-2retroviruses have been described in European patent application No.87,400,151,4 published under the number 239,425.

The HIV-1 retrovirus is the most common and its presence is predominantin several regions worldwide. As regards the HIV-2 retrovirus, it ismost often found in West Africa, although its propagation outside thisregion has recently been documented by Grez et al., (1994) J. Virol. 68,2161-2168.

The totality of primate immunodeficiency lenti-viruses, comprising thetype 1 and type 2 human immuno-deficiency viruses as well as severaltypes of non-human primate viruses, is increasing in size andcomplexity. The most common of these viruses, HIV-1, is currentlyspreading in the form of a worldwide epidemic and is responsible for amajor public health problem. Shortly after the identification andmolecular characterization of this virus, it was recognized as beinghighly variable, and currently comprises several subtypes (Myers, 1994,Louwagie, et al. 1993, Louwagie et al. 1992, Myers, G. (1994) HIV-1subtypes and phylogenetics trees. In: Human Retrovirus and AIDS 1994;Myers, G., Korber, B., Wain-Hobson, S., Smith, R. F. and Pavlakis, G.N., Eds. Los Alamos National Laboratory, Los Alamos, N. Mex.III-2-III-9.). This differentiation of subtypes is mainly based on thedivergence of the gag and env genes. At least 6 subtypes have beenidentified, designated A to F, but several are still likely to emergefrom the ongoing extensive worldwide survey on the isolates of HIV-1. Ithas been found that these various subtypes are equidistant from eachother, in a phylogenetic profile termed star phylogeny, which suggeststhat the various HIV-1 subtypes might have evolved and divergedsynchronously from a common ancestor.

Recently, two separate viruses of this group of HIV-1 viruses wereisolated and characterized. These two viruses were obtained frompatients living in Cameroon, in West Central Africa (Gürtler, et al.1994, Vanden Heasevelde, et al. 1994). Their sequence, more particularlythe sequence of their env (envelope) gene, shows clearly that theseviruses belong to a separate category of HIV-1-related viruses, referredto as HIV-1 group O (Nkengasong et al., 1993).

However, the diversity of the isolates within this group ofHIV-1-related viruses is not known, and its propagation outside Africahas not been documented.

A general constraint, in the development of HIV serological tests, is toavoid both falsely positive—or falsely negative—results while at thesame time retaining or improving the sensitivity in terms of detectionof seropositivity which the previous tests allow.

Tests based on the use of consensus peptide(s), essentially derived fromthe “env” gene, were considered as an almost ideal solution until thediscovery of the HIV-1-O variant brought to light the possibility offalsely negative results (Genomic cloning and complete sequence analysisof a highly divergent African human immunodeficiency virus isolate. J.Virol. 1994; 68: 1586-96; a new subtype of human immunodeficiency virustype 1 (MDV-5180) from Cameroon. J. Virol. 1994; 68: 1581-85).

The non-reactivity of certain tests with “env” peptide antigen, inpatients nonetheless exhibiting certain clinical syndromescharacteristic of AIDS or lymphadenopathy syndromes which occasionallyprecede them, is, at the present time, occasionally attributed to aninfection of the HIV-1-O group (HIV-1/HIV-2 seronegativity in HIV-1subtype O infected patients, Lancet 1994; 343: 1393-94; New HIV-1subtype in Switzerland. Lancet 1994; 344: 270-71).

BRIEF DESCRIPTION OF THE DRAWINGS

This invention will be more fully described with reference to thedrawings in which:

FIGS. 1A and B depict RT activity and p24 levels.

FIG. 2 is a Western blot analysis of a series of serum samples.

FIGS. 3A-D show an amino acid alignment of the HIV-1VAU and HIV-1LAIenvelope sequences (SEQ ID NO:102 and SEQ ID NO:103), including analignment of HIV-1VAU and HIV-1LAI gp120 sequences (SEQ ID NO:46 and SEQID NO:48) and HIV-1VAU and HIV-1LAI gp41 sequences (SEQ ID NO:47 and SEQID NO:49).

FIG. 4 is a multiple alignment of the immunodominant peptides in theextracellular segment of the transmembrane envelope glycoprotein ofvarious HIV-1 isolates (SEQ ID NOs:50-62).

FIGS. 5A-B contain branches of a phylogenetic tree.

FIG. 6 is the DNA sequence of HIV-1VAU env gene (SEQ ID NO:63). Shortnucleotide sequences located at the top of the figure are disclosed asresidues 1-12 and 2,620-2,631 of SEQ ID NO: 63.

FIG. 7 is the DNA sequence of HIV-1VAU virus integrase gene (SEQ IDNO:64).

FIGS. 8A-C contain a comparison of the GAG amino acid sequence ofvarious HIV-1 strains (SEQ ID NOs:65-70).

FIG. 9A is a multiple alignment of the V3 loop of gp120 (SEQ ID NOS 71,73, 75, 77, 79, 81, 83, 85, 87, 89, 93, 91, and 93, respectively, inorder of appearance).

FIG. 9B is a multiple alignment of the immunodominant region of gp41(SEQ ID NOS 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 94, 92, and 94,respectively, in order of appearance).

FIG. 10A is a 513 base pair DNA sequence of the GAG region of HIV-1 DUR(SEQ ID NO:95).

FIG. 10B is the corresponding 171 amino acid sequence of FIG. 10A (SEQID NO:96).

FIG. 11A is a 525 base pair DNA sequence of the V3 loop region of gp120of HIV-1DUR (SEQ ID NO:97).

FIG. 11B is the corresponding 175 amino acid sequence of FIG. 11A (SEQID NO:98).

FIG. 12A is a 312 base pair DNA sequence of the immunodominant region ofgp41 of HIV-1 DUR (SEQ ID NO:99).

FIG. 12B is the corresponding 104 amino acid sequence of FIG. 12A (SEQID NO:100).

FIG. 13A shows specific primer sequences for HIV-1 type O (SEQ IDNOs:42-45).

FIG. 13B shows the corresponding positions of the primers of FIG. 13A indifferent HIV type O strains.

FIGS. 14A-B are serological correlation results.

FIGS. 15A-C are nucleotide comparisons of HIV sequences.

FIGS. 16A-C are protein comparisons of HIV sequences.

FIG. 17 is a phylogenetic tree.

FIG. 18A-B show an HPLC chromatogram of a VAU peptide.

FIG. 19A-B show a mass spectroscopy analysis of a VAU peptide (SEQ IDNO:101).

FIGS. 20A-C contain immunoreactivity data.

FIGS. 21A-C contain immunoreactivity data.

DESCRIPTION OF THE INVENTION

The aim of the present invention is to provide diagnostic laboratorieswith means, in particular specific peptides, allowing a detection ofanti-HIV antibodies which were hitherto liable to be undetected. Theinvention also relates to mixtures of peptides obtained from HIV-1 DURand of corresponding peptides from other HIVs, so as to avoid potential“false negative” results.

The invention moreover relates to a process of detection and ofdiscrimination, in a biological sample, between antibodiescharacteristic of an HIV-1-M type retrovirus and antibodiescharacteristic of an HIV-1 group (or subgroup) O retrovirus.

The invention stems from observations made on a seropositive woman whohad stayed in the Cameroon and who had revealed an atypical serologicalreactivity, in the course of several tests of screening for HIVinfection, these tests having been confirmed by “Western blot”techniques.

On account of this atypical serological reactivity, in particular thelack of reactivity to certain third-generation tests, even modified forthe O type, the inventors considered it interesting to carry outsequencing of certain parts of the genome of this HIV-1 DUR strain, morespecifically of the GAG and ENV genes.

However, gene amplifications by PCR using primers obtained from the Mgroup and known primers from the O group were unsuccessful for the partscoding for the V3 loop of gp120, and for the immunodominant region ofgp41. Only the GAG region could be amplified using primers known in theprior art (Loussert-Ajaka I, Lancet 1994; 343: 1393). Another aim of thepresent invention is consequently to determine primers capable ofovercoming this problem.

Partial sequences of the glycoproteins gp41 and gp120 were determined,along with capsid proteins (GAG gene), from lymphocyte DNA and fromviral cultures, indicating that this HIV-1 DUR strain belongs in part tothe HIV-1-O group, and that it differs considerably from the M group,more particularly as regards gp41 and gp120.

Thus, it was possible to demonstrate, more particularly as regards theGAG sequence of HIV-1 (DUR), the existence of consensus sequences in theO group, in several regions, which are distinct from the consensussequences of the M group in the same regions.

Cloning of the sequences coding for the GAG, gp41 and gp120 fragments ofHIV-1_((DUR)) was carried out in a Bluescript® plasmid containing a PST1site. The amplification products were cloned either according to thestandard techniques using T3 and T7 universal primers, or directlysequenced by using the primers of the preceding amplification. Thesequences were then determined with the Applied Biosystems 373Aautomated sequencer (ESGS Montigny le Bretonneux, France).

Within the context of the present invention, the inventors have isolatedand sequenced the env gene from an O group isolate, HIV-1_((VAU)),obtained from a French patient who had never traveled outside Europe andwho died of AIDS in 1992. According to its envelope sequence,HIV-1_((VAU)) is related to two recently characterized Cameroonianviruses HIV_(ANT70) and HIV_(MVP5180) Phylogenetic analysis of the envsequences reveals that the three viruses appear to constitute a separategroup, which will be referred to herein as HIV-1 group O. The isolationof HIV-_((VAU)) from this patient also indicates that a degree ofpropagation of HIV-1 group O has already occurred outside Africa.

Isolation of the HIV-1_((VAU)) Virus

HIV-1_((VAU)) was isolated in 1992 from a 41-year-old French patientsuffering from AIDS. This patient exhibited, in 1986, a severeleuconeutropenia associated with a carcinoma of the uterine cervix.However, she gradually showed signs of opportunistic infections, with areduction in the number of circulating CD4⁺ T cells and she died of AIDSin 1992. Anti-HIV-1 antibodies were first detected by ELISA (Elavia,Sanofi Diagnostics Pasteur and Abbott test) in 1990.

The patient had never traveled outside Europe, had not used intravenousmedicinal products and had not received any known blood transfusion. Nosexual partner of African origin has been identified. She gave birth toa healthy child in 1971, but a son, born in 1980, died at the age of onefollowing a clinical episode highly suggestive of neonatal AIDS. Herthird child, born in 1983, and her husband are currently in good healthand not infected.

The isolation of the virus was carried out in the following manner: theCD8⁺ cells present in the PBMCs (peripheral blood lymphocytes) of thepatient were removed using beads coated with IOT8 antibodies(Immunotech). These remaining PBMCs were stimulated with PHA, thencocultured with CD8-depleted PBMCs obtained from a healthy donor andstimulated with PHA. Viral growth in the coculture was monitored byassaying the reverse transcriptase (RT) activity of the supernatant andby ELISA test of the HIV-1 p24 (diagnostic kit marketed by DuPont deNemours). The virus obtained from the initial coculture was subjected toseveral passages in CD8-depleted and PHA-stimulated PBMC cultures.Several attempts were made to infect, with the HIV-1_((VAU)) varioustransformed cell lines, including MT4 cells (Harada, et al. 1985) andCEM cells (Rey, et al. 1989), as well as the Hela-CD4-LTRLacZ cell lineP4-2 (Clavel and Chameau 1994).

Biological Characterization of HIV-1_((VAU))

Two weeks after coculturing the patient's CD8-depleted, PHA-stimulatedPBMCs with similar bells from a healthy donor, the production of viruswas detected in the form of an RT activity peak in the culturesupernatant. This virus could then be subjected to serial passages onCD8-depleted, PHA-stimulated normal PBMCs. FIG. 1A represents theproduction of HIV-1_((VAU)) in infected PBMC culture supernatants,checked by RT assay (filled circles) and HIV-1 p24 antigen capture ELISA(empty circles). The concentration of HIV-1 p24 is expressed in ng/mland the RT activity in cpm/μl. In FIG. 1B, the same experiment wascarried out with a standard primary HIV-1 isolate from an AIDS patient.

Although the growth of HIV-1_((VAU)) was easily detected by RT assay,the detection of virus in the culture supernatants by HIV-1 p24 ELISA(DuPont) was substantially less sensitive. FIG. 1 shows the comparisonbetween the profiles of productive infection of PBMCs either withHIV-1_((VAU)) or with a primary HIV-1 isolate from an AIDS patient,assayed by RT or p24. For equivalent quantities of particles, determinedby assay of RT activity in the supernatants assayed, approximately 25times less p24 was detected in the case of HIV-1_((VAU)) than in thecase of the other HIV-1 isolate. The difference may be due to the factthat the monoclonal antibody specific for HIV-1 p24, which is used tocoat the ELISA plates, has only a weak affinity for the gag products ofHIV-1_((VAU)).

Several negative attempts were made to propagate HIV-1_((VAU)) ontransformed human T cell lines sensitive to HIV-1. In particular,cocultures between PBMCs infected with HIV-1_((VAU)) and either MT4cells or CEM cells did not lead to propagation of the virus. It was alsofound that this virus was not capable of infecting CD4⁺ HeLa cells(P4-2) (Clavel and Charneau 1994) carrying a lacZ gene inducible by thetat gene. Likewise, no replication of HIV-1_((VAU)) could be detected inactivated peripheral blood lymphocytes from several chimpanzees.

Analysis of the HIV-1_((VAU)) envelope sequence, which will be describedin detail later, and its comparison with that of the two recentlydescribed Cameroonian isolates indicate that all three viruses belong tothe same group of HIV-1-related viruses. Furthermore, this comparisonindicates that these three variants of the virus are approximatelyphylogenetically equidistant from each other. Consequently, each of thethree virus variants constitutes on its own a distinct subtype of theirgroup, which is now called HIV-1 group O. This group is different fromthe group of other HIV-1 isolates, identified up until now, which theinventors call here HIV-1, group M.

The appearance of this new group poses the question of its origin: didgroup O evolve from group M viruses (or conversely) or does each grouphave a different history? The inventors think that, insofar as bothgroup M and group O have a similar internal divergence profile, it islikely that they each correspond to the diversification of distinctviral ancestors in distinct human populations. It is not possible toassess from the phylogenetic and virological data currently availablewhether the ancestor of either of the two groups affected humansnaturally or was introduced into humans from other species. The onlyvirus similar to HIV-1 present in a nonhuman primate is the SIVCPZGA3isolate (Huet, et al. 1990), isolated from a chimpanzee apparentlyinfected naturally, which is clearly different both from group M andfrom group O, and for which no human equivalent has been found. It isunlikely that the group O viruses evolved recently from a chimpanzeevirus insofar as HIV-1_((VAU)) has not succeeded in replicating inchimpanzee lymphocytes.

Why does the group O epidemic appear only now, some 15 to 20 years laterthan group M? There are three possible explanations: firstly, theintroduction of the ancestor of the group O viruses into humans isthought to have occurred more recently than that of croup M; secondly,it is possible that group M was allowed to spread earlier compared withgroup C because of different social conditions in their region oforigin; and thirdly, the group O viruses could have a lower capacity fortransmission compared with that of the group M viruses. It has beenproposed that such a property explains the absence of significantworldwide propagation of HIV-2, for which a smaller viral load ininfected subjects is linked to reduced transmissibility (De Cock, et al.1993). In this regard, although no data are available on the viral loadin patients infected with an HIV-1 group O, the pathogenicity of theseviruses does not appear to be different from that of HIV-1. The patientfrom whom HIV-1_((VAU)) was isolated died of AIDS, like the patient fromwhom the HIV_(MVP5180) group O isolate was obtained.

However, the natural history of infection of the HIV-1_((VAU)), patientis still not clear, but there are several indications that this patientwas infected before 1980, as suggested by the death on that date of hersecond child suffering from a syndrome resembling AIDS.

The invention relates to any variant of the nucleic acid sequences ofthe HIV-1_((VAU)) virus or of any group O equivalent virus, containingstructural proteins which have the same immunological properties as thestructural proteins coded for by the env gene comprising the sequencedescribed in FIG. 6 and called “vau”, also designated by SEQ ID NO:63.

The present invention also relates to compositions containing eitherantigens according to the invention, or a mixture of antigens accordingto the invention combined with extracts originating from one or moreHIV-1 group O viruses or from other variant viruses, on the one hand,and from one or more HIV-2 and/or HIV-1 viruses, on the other hand,these compositions being optionally labeled. It is possible to use anytype of appropriate label: enzymic, fluorescent, radioactive, etc.

Nucleic Acids

The invention relates to the DNAs or DNA fragments, more particularlycloned DNAs and DNA fragments, obtained from RNA, cDNA or primers whichcan be used in PCR, or other gene amplification methods, derived fromthe HIV-1_((VAU)) retrovirus RNA or DNA. The invention relates moreparticularly to all the equivalent DNAs, especially to any DNA havingsequence homologies with the HIV-1_((VAU)) DNA, in particular with thesequence coding for the env region of the HIV-1_((VAU)) straincomprising the sequence corresponding to SEQ ID NO:63 represented inFIG. 6 and called “vau”. The homology with HIV-1 group M is at leastequal to 50%, preferably to 70% and still more advantageously to about90%. Generally, the invention relates to any equivalent DNA (or RNA)capable of hybridizing with the DNA or RNA of a group O HIV-1retrovirus.

The invention also relates to the RNA sequences corresponding to the DNAsequences defined above.

The invention also relates to the HIV-1_((VAU)) virus integrase genecomprising the sequence identified by the name SEQ ID NO:64 orhybridizing with SEQ ID NO:64. The invention also relates to the RNAscorresponding to the DNA described above.

The subject of the invention is also compositions containing thepeptides or polypeptides encoded by the abovementioned DNA or DNAfragments.

Oligonucleotides derived from the VAU sequence or alternatively from theHIV-1_((VAU)) virus integrase gene, particularly oligonucleotidescomprising at least 9 nucleotides, may be used for the detection ofgroup O HIV-1 virus DNA or RNA sequences in biological samples, cellcultures or cell extracts, by the PCR technique or any other geneamplification technique. These sequences could be used either as geneamplification primers or as probes for the specific detection of thegene amplification products. Also capable of being used as hybridizationprobes are the amplification products, or their corresponding syntheticsequence, obtained by chemical synthesis (Applied Biosystems).

The invention also covers any fragment of at least 100 nucleotides whichmay be used as a probe in hybridization reactions and capable ofpermitting reaction with part of the genome of an HIV-1_((VAU)) variantunder high stringency hybridization conditions.

Cloning and Sequencing of the HIV-1_((VAU)) env Gene

For the initial PCR amplification of the HIV-1_((VAU)) DNA, the totalDNA was extracted from PBMCs infected with HIV-1_((VAU)) and a segmentof the pol gene (integrase region) was amplified using degenerateprimers:

Seq. ID No. 1 primer 4506: 5′AGTGGAT(A/T) (T/C)ATAGAAGCAGAAGT3′;; Seq.ID No. 2 primer 5011: 5′ACTGC(C/T)CCTTC(A/C/T)CCTTTCCA3′;;

The reaction medium including 50 mM KCl, 10 mM Tris-HCl (pH 8.9), 1.5 mMMgCl₂, 0.1 mg/ml gelatin, 0.2 mM dNTP, 1U of Taq polymerase (Amersham).The PCR was carried out in 43 thermal cycles at 92° C. for 10 seconds,50° C. for 1 minute and 72° C. for 40 seconds.

The resulting amplification product was cloned into a pBluescript™vector, generating the clone ph4, deposited at the Collection Nationaledes Cultures de Micro-organismes (CNCM), Institut Pasteur, 28 rue duDocteur Roux, 75724 Paris Cedex France, on 20 Oct. 1994 under No.I-1486, which was subsequently used as a probe to screen a lambdalibrary of low molecular weight DNA, which was digested with EcoRl andwas obtained from cells infected with HIV-1_((VAU)). Briefly, the PBMCsinfected with HIV-1_((VAU)) were cocultured for 24hours with new PBMCsstimulated with PHA and depleted of CD8⁺cells, after which a highcytopathic effect (CPE) was visible. The low molecular weight DNA wasthen extracted according to the Hirt method (Hirt 1967), and digestedwith the enzyme EcoRI. A previous Southern-blot analysis of this DNA hadindeed shown that the HIV-1_((VAU)) genome contained only one EcoRIsite, permitting the cloning of nonintegrated circular DNA speciesrepresenting the entire viral genome. The resulting digestion productwas subjected to agarose gel electrophoresis, and the population of DNAfragments of approximately 8-12 kb in size was purified and ligated toEcoRl-digested lambda Zap DNA™ (Stratagene). After encapsidation,plating and screening by hybridization with ³²P-labeled ph4 DNA, aclone, lamda H34, was identified as being positive, and amplified. TheEcoRI insert was purified, sonicated, and cloned by the “shotgun”technique into the phosphatase-treated vector M13 mp18 digested with theenzyme SmaI. One hundred and fifty of the clones obtained were sequencedin a 373A DNA sequencer (Applied Biosystems), and the resultingsequences were assembled into a single sequence using the Wisconsin GCGDNA analysis package.

Analysis of this sequence revealed numerous nonsense codons in all thereading frames, which is highly suggestive of a hypermutated genome(Vartanian, et al. 1991). This sequence being unusable, it wasconsequently decided to amplify, by PCR, the HIV-1_((VAU)) env geneusing the total DNA from PBMCs infected with HIV-1_((VAU)), and theoligonucleotide primers derived from the sequence λH34:

primer TH2 5′GCTCTAGATGGGGATCTCCCATGGCAGG3′; Seq. ID No. 3 primer UH25′GCTCTAGATCAGGGAAGAATCCCTGAGTGT3′.. Seq. ID No. 4

The PCR amplification was carried out in 35 thermal cycles at 92° C. for15 seconds, 52° C. for 1 minute, 60° C. for 2 minutes and 72° C. for 2minutes. The resulting amplification product, of 3.5 kb in size, wascloned into the M13 mp18 vector and sequenced by successive reactions,first using the M13 universal sequencing primer, and then the primersdeduced from the upstream sequences. Analysis of the nucleotide andpeptide sequences was carried out using the Wisconsin GCG DNA analysispackage. The HIV-1_((VAU)) env gene codes for 877 amino acids in total,including the signal peptide. The nucleotide sequence of theHIV-1_((VAU)) env gene corresponds to Seq. ID NO:63 (see FIG. 3).

Use of Nucleic Acids as Probes

The invention also relates naturally to the use of DNA, cDNA orfragments thereof, or of recombinant plasmids or other equivalentvectors containing these fragments, as probes, for detecting thepresence or otherwise of the HIV-1_((VAU)) virus in serum samples orother biological fluids or tissues obtained from patients suspected ofbeing carriers of the HIV-1_((VAU)) virus. These probes are optionallylabeled (radioactive, enzymic or fluorescent labels and the like).Probes which are particularly valuable for the implementation of themethod for detecting the HIV-1_((VAU)) virus or an HIV-1_((VAU)) variantmay be characterized in that they comprise all or a fraction of the DNAcomplementary to the HIV-1_((VAU)) virus genome or alternativelyespecially the fragments contained in various clones. An HIV-1_((VAU))cDNA fraction containing all or part of the env region will be mentionedmore particularly.

The probes used in this method for detecting the HIV-1_((VAU)) virus orin diagnostic kits are not in any way limited to the probes describedpreviously. They comprise all the nucleotide sequences obtained from thegenome of the HIV-1_((VAU)) virus, an HIV-1_((VAU)) variant or a virussimilar by its structure, provided that they allow the detection, usingbiological fluids from individuals likely to have AIDS, of an HIV-1group O virus, in particular HIV-1_((VAU)) by hybridization with theHIV-1_((VAU)) virus DNA or RNA.

Particularly advantageous are the probes which, when hybridized withHIV-1, give a strong reaction with HIVs belonging to group O and a weakreaction with HIVs belonging to group M. By way of nonlimiting example,a probe constructed from the HIV-1_((VAU)) virus integrase gene sequence(SEQ ID NO:64) gives, when it is hybridized with HIV-1 underhybridization conditions such as those described in Patent EP 178 978, astrong reaction with group O HIVs and a weak reaction with group M HIVs.

The detection may be performed in any manner known per se, especially:by bringing these probes into contact either with the nucleic acidsobtained from cells contained in biological fluids (for example spinalfluid, saliva and the like), or with these fluids themselves, providedthat their nucleic acids have been made accessible to hybridization withthese probes, and this under conditions permitting hybridization betweenthese probes and these nucleic acids, and by detecting the hybridizationwhich maybe produced.

The abovementioned diagnosis involving hybridization reactions may alsobe performed using mixtures of probes derived from HIV-1_((VAU)), HIV-1and HIV-2 respectively, provided that it is not necessary to make adistinction between the desired HIV virus types.

The subject of the invention is also expression vectors containing thesequence coding for the HIV-1 envelope proteins or containing thesequence coding for the integrase.

The invention comprises compositions for detecting the presence orotherwise of the HIV-1_(VAU) virus in serum samples or samples of otherbiological fluids or tissues, obtained from patients likely to becarriers of the HIV-1_(VAU) virus. These compositions are characterizedin that they comprise at least one probe obtained from a nucleotidesequence obtained or derived from the HIV-1_(VAU) virus genome,particularly an HIV-1_(VAU) DNA fragment containing the region or partof the region coding for the env protein of the HIV-1_(VAU) virus or ofan HIV-1 VAU variant.

Advantageously, the composition described above also comprises a probeobtained from a nucleotide sequence derived from HIV-1 or HIV-2.

Other diagnostic compositions comprise the primers of the inventionwhich are capable of being used in gene amplification of subgroup Oretroviruses or variants of these retroviruses.

Antigens, Especially Proteins and Glycoproteins

The invention relates to an HIV-1 group (or subgroup) O retroviralprotein, or natural or synthetic peptide or polypeptide comprising atleast a part of said protein, which is capable of being recognized byantibodies which may be isolated from serum obtained after an infectionwith an HIV-1 group O VAU strain, or an HIV-1 group O DUR strain.

The invention relates to an external envelope protein of the HIV-1_(VAU)retrovirus encoded by the gene comprising the sequence corresponding toSEQ ID NO:63. According to a preferred embodiment of the invention, thisprotein is in addition characterized in that it comprises the amino acidsequence corresponding to SEQ ID NO:46 represented in FIG. 3 andcomprising amino 20 acid residues 1 to 526. The subject of the inventionis also any polypeptide or variant which is derived from said sequencehaving an epitope which may be recognized by antibodies induced by theHIV-1_(VAU) virus.

The abovementioned protein may be obtained in a glycosylated ornonglycosylated form.

The subject of the invention is also an envelope transmembrane proteincomprising the amino acid sequence SEQ ID NO:47 represented in FIG. 3between amino acid residues 527 and 877. This transmembrane protein is,within the scope of the invention, in glycosylated or nonglycoslatedform.

The invention relates to all the antigens, especially proteins,glycoproteins, polypeptides or peptides, obtained by expressing codingsequences of the HIV-1_((VAU)) genome and having immunologicalproperties equivalent to those of HIV-1_((VAU)). The antigens are saidto be equivalent within the scope of the present invention provided thatthey are recognizable by the same antibodies, especially antibodieswhich can be isolated from serum obtained from a patient who has beeninfected with an HIV-1_((VAU)).

In particular, the subject of the invention is the peptides orpolypeptides which are synthesized chemically and whose amino acidsequence is contained in that of the HIV-1_((VAU)) envelope proteins,which sequence is represented in FIG. 3, or the equivalent peptides orpolypeptides.

There should also be included among the equivalent peptides,polypeptides, proteins or glycoproteins, the fragments of the aboveantigens and the peptides which are prepared by chemical synthesis or bygenetic engineering, so long as they give rise to immunologicalcross-reactions with the antigens from which they are derived. In otherwords, the invention relates to any peptide or polypeptide havingepitopes which are identical or similar to the epitopes of theabovementioned antigens and which are capable of being recognized by thesame antibodies. Forming part of this latter type of polypeptides arethe products of expression of DNA sequences corresponding to DNAsequences coding for the polypeptides or antigens mentioned above.

More particularly, the antigens which are obtained from theHIV-1_((VAU)) virus or produced by genetic engineering or conventionalchemical synthesis, and which are of the greatest interest within thecontext of the present invention, are the antigens which make itpossible to obtain a clear distinction between the HIV-1_((VAU)) virusesof the invention and the viruses of the HIV-1 and HIV-2 groups. In thisregard, considerable differences have been observed at the level of theHIV-1_((VAU)) virus envelope protein as well as at the level of theimmunodominant epitope of the external portion of the PM protein. Itappears that the gag and pol proteins exhibit greater similarity withthe HIV-1 virus than the envelope protein.

The invention also relates to peptides or polypeptides which areidentical to the immunodominant region of the HIV-1_((VAU)) envelopetransmembrane glycoprotein. This region is represented in FIG. 3.

Preferred polypeptides of this region are, for example, those whichcontain the sequence CKNRLIC (SEQ ID NO:5) or correspond to thissequence. They may also be peptides or polypeptides corresponding to thesequence RLLALETFIQNWWLLNLWGCKNRLIC (SEQ ID NO:6) or comprising thissequence.

Another preferred peptide, identified below by the name “VAU peptide”,corresponds to the following sequence or comprises this sequence or anypart of this sequence capable of being recognized by antibodies directedagainst the HIV-1_((VAU)) retrovirus

(SEQ ID NO: 7) RARLLALETFIQNQQLLNLWGCKNRLICYTSVKWNKT.

Variant polypeptides of this sequence are for example the polypeptidesrepresented in FIG. 4 for the HIV-_((MVP5180)) and HIV-1_((ant70))isolates. These polypeptides may also be derived from the preceding onesby insertion and/or deletion and/or substitution, for exampleconservative substitution by amino acid residues.

The present invention relates obtained from the HIV-1-O DUR virusdeposited on 23 Feb. 1995 at the Collection Nationale des Cultures deMicro-organismes (CNCM), Institut Pasteur, 28 rue du Docteur Roux, 75724Paris Cedex France, under the reference I-1542, or a peptide whosesequence is distinguished from that of the above by substitution,deletion or addition of amino acids, this separate peptide neverthelessretaining the antigenic characteristics of the above one.

Other peptides falling within the scope of the invention are definedbelow.

Thus, a preferred peptide of the invention is a peptide containing atleast 4 consecutive amino acids contained in the GAG sequencerepresented in FIG. 8A-C or in an immunologically similar GAG sequenceobtained from a variant of the HIV-1-O DUR virus, said immunologicallysimilar sequence being recognized by antibodies which also specificallyrecognize at least one of the sequences AHPQQA (SEQ ID NO:8), LWTTRAGNP(SEQ ID NO:9) contained in the GAG sequence of FIG. 8.

Preferably, this peptide consists of a peptide whose amino acid sequenceis contained either in one of the following sequences:

(SEQ ID NO: 10) SPRTLNAWVKAVEEKAFNPEIIPMFMALSEGA (1) (SEQ ID NO: 11)MLNAIGGHQGALQVLKEVIN (2) (SEQ ID NO: 12) GPLPPGQIREPTGSDIAGTTSTQQEQI (3)(SEQ ID NO: 13) IPVGDIYRKWIVLGLNKMVKMYSPVSILDI (4) (SEQ ID NO: 14)QGPKEPFRDYVDRFYKTKLAE (5) (SEQ ID NO: 8) AHPQQA (5a) (SEQ ID NO: 9)LWTTRAGNP (5b)or in a corresponding immunologically similar sequence, this peptidecontaining at least 4 consecutive amino acids of one of said sequences.

Preferably also, this peptide consists of a peptide whose amino acidsequence is contained either in one of the following sequences:

SPRTLNAWVK (6) (SEQ ID NO: 15) GSDIAGTTST (7) (SEQ ID NO: 16)QGPKEPFRDYVDRF (8) (SEQ ID NO: 17)or in a corresponding immunologically similar sequence, this peptidecontaining at least four consecutive amino acids of one of saidsequences.

Peptides which are particularly preferred in the present invention arethe peptides containing:

-   -   the amino acid sequence NPEI (9) (SEQ ID NO: 18) or    -   the amino acid sequence AVEEKAFNPEIIPMFM (10) (SEQ ID NO: 19),        and more particularly peptides whose amino acid sequence is        contained, either in one of the following sequences:

IGGHQGALQ (23) (SEQ ID NO: 20) REPTGSDI (24) (SEQ ID NO: 21)or in a corresponding immunologically similar sequence, this peptidecontaining at least 4 consecutive amino acids of one of said sequences,as well as the peptide whose amino acid sequence is contained, in thefollowing amino acid sequence:

-   -   INDEAADWD (25) (SEQ ID NO: 22)        or in a corresponding immunologically similar sequence, this        peptide containing at least 4 consecutive amino acids of said        sequence.

IDEAADWD (25) (SEQ ID NO: 22)or in a corresponding immunologically similar sequence, this peptidecontaining at least 4 consecutive amino acids of said sequence.

The present invention relates to the nucleic acid sequences coding forpeptides (23), (24) and (25) as well as the nucleic acid sequencescoding for the immunologically similar sequences, as well ascompositions comprising at least one of these nucleic acids.

The invention also relates to the use of at least one of these nucleicacids for detection and discrimination between HIV-1 group M and HIV-1group O strains. A peptide derived from the HIV-1-O DUR virus definedabove also falls within the scope of the present invention, said peptidecontaining at least 4 consecutive amino acids of the V3 loop of gp120represented in FIG. 9A or of 25 immunologically similar sequence,variant of the HIV-1-O DUR virus, said immunologically similar sequencebeing recognized also specifically recognize by antibodies which alsospecifically recognize at least one of the sequences:

KEIKI (12), (SEQ ID NO: 23) EREGKGAN (13), (SEQ ID NO: 24)CVRPGNNSVKEIKI (14), (SEQ ID NO: 25) QIEREGKGANSR (15). (SEQ ID NO: 26)

This peptide preferably contains:

a) either the sequence CVRPGNNSVKEIKIGPMAWYSMQIEREGKGANSRTAFC (11) (SEQID NO:27) or a part of this sequence which contains at least 4 aminoacids

b) or an amino acid sequence which is separate from the sequence of a)in which one or more amino acids are replaced with two amino acids, withthe proviso that the peptide retains its reactivity with an antiserumagainst the abovementioned peptide,

c) or an amino acid sequence which is separate from a) or b), in whichone or more amino acids have been deleted or added, with the provisothat the peptide retains its reactivity with an antiserum against thepeptide of a),

d) or a corresponding immunologically similar sequence or part ofsequence.

Preferably also, this peptide contains either

the sequence KEIKI (12) (SEQ ID NO:23), or

the sequence EREGKGAN (13) (SEQ ID NO:24), or

the sequence GPMAWYSM (16) (SEQ ID NO:28).

In a particularly preferred manner, a peptide as defined above containsthe amino acid sequence

CVRPGNNSVKEIKI (14) (SEQ ID NO:25) or the sequence

QIEREGKGANSR (15) (SEQ ID NO:26).

A peptide derived from the HIV-1-O DUR virus as defined above also fallswithin the scope of the invention, said peptide containing at least 4consecutive amino acids, whose entire sequence is contained in thesequence of the immunodominant region of gp41 represented in FIG. 9 orin a corresponding immunologically similar sequence, obtained from avariant of the HIV-1-O DUR virus, said immunologically similar sequencebeing recognized by antibodies which also specifically recognize atleast one of the following sequences:

(SEQ ID NO: 29) RLLALETLMQNQQL (17), (SEQ ID NO: 30)LNLWGCRGKAICYTSVQWNETWG (18), (SEQ ID NO: 31) CRGKAI (19), (SEQ ID NO:32) SVQWN (20), (SEQ ID NO: 33) RLLALETLMQNQQLLNLWGCRGKAICYTS (21), (SEQID NO: 34) QNQQLLNLWGCRGKAICYTSVQWN (22).

This peptide is preferably a peptide containing the sequence

RLLALETLMQNQQL (17) (SEQ ID NO:29), or

LNLWGCRGKAICYTSVQWNETWG (18) (SEQ ID NO:30)

or part of this peptide (18) (SEQ ID NO:30) containing:

-   -   a) either the sequence CRGKAI (19) (SEQ ID NO:31) or the        sequence SVQWN (20) (SEQ ID NO:32) in which Q is, where        appropriate, replaced by a different amino acid, which is        nevertheless also different from K, or the two sequences at the        same time,    -   b) or an amino acid sequence which is separate from the sequence        of a) in which one or more amino acids are replaced with two        amino acids, with the proviso that the peptide retains its        reactivity with an antiserum against the peptide of a),    -   c) or an amino acid sequence which is separate from a) or b), in        which one or more amino acids have been deleted or added, with        the proviso that the peptide retains its reactivity with an        antiserum against the peptide of a),    -   d) or in a corresponding immunologically similar sequence or        part of sequence.

Preferably also, this peptide possesses one or the other of thefollowing characteristics:

-   -   its N-terminal sequence which contains at least 8 amino acids is        not immunologically recognized by antibodies formed against the        sequence RILAVERY (SEQ ID NO:35) contained in the immunodominant        region of gp41 of the 35 HIV-1-LAI strain.    -   it is not recognized by antibodies formed against the peptide        SGKLIC (SEQ ID NO:36) of the HIV-1-LAI strain.    -   it contains either of the following two sequences:

(SEQ ID NO: 33) RLLALETLMQNQQLLNLWGCRGKAICYTS (21) (SEQ ID NO: 34)QNQQLLNLWGCRGKAICYTSVQWN (22).Synthesis of VAU Peptides

A VAU peptide was prepared by the conventional solid phase peptidesynthesis technique using the “continuous flow” Fmoc method. The peptidewas prepared using a Milligen 9050 PEP synthesizer and using the“Millipore” PEG PAL resin, substituted with the first C-terminal aminoacid residue. The side chains of the amino acids are protected by thefollowing groups: Pmc for arginine; Trt for asparagine, glutamine andcysteine; Boc for lysine; tBu ester for glutamic acid; tBu ether forserine, threonine and tyrosine. The temporary Fmoc groups are removedwith a 20% piperidine solution in DMF. The reactions for coupling eachamino acid are performed with 6 equivalents of DIPCDI and HOBT. Someresidues require a double coupling especially arginines 1 and 23,cysteines 19 and 26, asparagine 11, glutamines 10, 12 and 13, alanine 4,isoleucine 9 and leucines 2, 3, 14 and 15.

After coupling, the resin is dried under vacuum. The peptide is cleavedfrom the support by the K reagent for 4 hours at room temperature. Thecrude peptide is precipitated and washed with ethyl ether. The productis purified by high-pressure liquid chromatography (HPLC) in a WATERS LCPREP 4000 instrument with WATERS Delta Pak C1 8 40×100 mm cartridges,flow rate 30 ml/min, acetonitrile/0.1% TFA gradient. The fractionscontaining the peptide are combined, concentrated in a rotary evaporatorand then lyophilized.

Cyclization

The peptide (0.025 mM) is dissolved in a 10 mM ammonium acetatesolution. The pH is adjusted to 8.5 with 1 M ammonium hydroxidesolution. The pH is readjusted after 3 or 4 hours. The cyclization ismonitored by HPLC at 214 nm and 280 nm, WATERS Delta Pak C18 5μ column,acetonitrile/0.1% TFA gradient. The cyclization is complete after 15hours. The pH is brought to 6 using 97-100% acetic acid, the solution islyophilized and then purified under the same conditions as for the crudepeptide.

The peptide is checked by HPLC and by mass spectrometry according to theelectrospray technique (FIGS. 18A-B and FIGS. 19A-B) (FISON VG Trio 2000spectrophotometer).

Fmoc: 9-Fluoraenylmethyloxycarbonyl Pmc:8-Methylpentane-6-sulfonylchroman Trt: Tritryl Boc: TertbutyloxycarbonyltBU: tert butyl DMF: Dimethylformamide DIPCDI: DiisopropylcarbodiimideHOBT: 1-Hydroxybenzotriazole TFA: Trifluoroacetic acid Reagent K:Phenol/water/thioanisole/ethanedithiol/TFA; 2.5 ml/2.5 ml/ 2.5 ml/1.5ml/41 ml

Comparison of the amino acid sequence of the HIV-1_((VAU)) envelope withthe corresponding sequence of other HIV viruses.

Western-blot analysis of a series of serum samples obtained from apatient infected with HIV-1_((VAU)) is presented in FIG. 2.Nitrocellulose strips, carrying proteins separated by electrophoresisand obtained from purified HIV particles (LAV BLOT™, SANOFI DIAGNOSTICSPASTEUR), were incubated with serum samples and their reactivity wasevaluated according to the procedures recommended by the manufacturer.The results obtained are the following: strip 1: proteins specific forHIV-2, which have been reacted with serum sample obtained in February1992 from the HIV-1_((VAU)) patient. Strips 2-7: HIV-1 positive sera;sera from the HIV-_(( VAU)) patient: 2: obtained in November 1990; 3: inDecember 1990; 4: in February 1991; 5: in February 1992; 6: negativecontrol; 7: positive control (serum from an individual infected withHIV-1). The names and the sizes of the proteins (in kD) are indicated inthe margin.

FIG. 3 shows an alignment of the amino acid sequence of theHIV-1_((VAU)) envelope with the corresponding sequence of the HIV-1-LAIreference isolate (Wain-Hobson, et al. 1985). The signal peptides, theV3 loop and the gp41 immunodominant epitope are highlighted by shadedrectangles. The site of cleavage between the external envelopeglycoprotein gp120 and the transmembrane gp41 is indicated by arrows.The vertical lines between the amino acid letters indicate completeidentity, colons (:) indicate high homology, and dots (.) indicatelimited homology between individual amino acids. The alignment wasperformed using the GAP program of the Wisconsin GCG package.

The original version (1.0) of the GAP and BESTFIT programs was writtenby Paul Haeberli from a detailed study of the publications of Needlemanand Vunsch (J. Mol. biol. 48, 443-453 (1970) and of Smith and Waterman(Adv. Appl. Math. 2; 482-489 (1981). The limited alignments weredeveloped by Paul Haeberli and added to the package to constitute the3.0 version. They were then fused into a single program by PhilipMarquess to constitute the 4.0 version. The gap absence penalties in thealignment of the proteins were modified as suggested by Rechid, Vingronand Argos (CABIOS 5; 107-113 (1989)).

The alignment of FIG. 3 shows numerous regions of high divergence, witha few domains retained here and there. These retained regions correspondroughly to the domains also retained in the conventional HIV-1 isolates(Alizon et al. 1986, Benn et al. 1985). Among the divergent domains, theV3 loop, also called principal determinant of neutralization (Javaherianet al. 1990, Javaherian et al. 1989, Matsushita et al. 1988) is clearlyone of the most divergent, although the two cysteines defining the loopare retained. The sequence of the cap of the loop, GPGRAF (SEQ ID NO:37)for HIV-1-LAI is GPMAWY (SEQ ID NO:38) in HIV-1_((VAU)). This unit ofthe cap is identical to that of the Cameroonian group O isolateHIV_((ANT70)) (Van den Heasevelde et al. 1994), but is different fromthat of the other group O isolate, HIV_(MVP5180) (Gürtler et al. 1994),for which the motif is GPMRWR (SEQ ID NO:39).

A VAU peptide was prepared by the conventional solid phase peptidesynthesis technique using the “continuous flow” Fmoc method. The peptidewas prepared using a Milligen 9050 PEP synthesizer™ and using the“Millipore” PEG PAL™ resin, substituted with the first C-terminal aminoacid residue. The side chains of the amino acids are protected by thefollowing groups: Pmc for arginine; Trt for asparagine, glutamine andcysteine; Boc for lysine; tBu ester for glutamic acid; tBu ether forserine, threonine and tyrosine. The temporary Fmoc groups are removedwith a 20% piperidine solution in DMF. The reactions for coupling eachamino acid are performed with 6 equivalents of DIPCDI and HOBT. Someresidues require a double coupling especially arginines 1 and 23,cysteines 19 and 26, asparagine 11, glutamines 10, 12 and 13, alanine 4,isoleucine 9 and leucines 2, 3, 14 and 15.

After coupling, the resin is dried under vacuum. The peptide is cleavedfrom the support by the K reagent for 4 hours at room temperature. Thecrude peptide is precipitated and washed with ethyl ether. The productis purified by high-pressure liquid chromatography (HPLC) in a WATERS LCPREP 4000™ instrument with WATERS Delta Pak C1™ 8 40 ×100 mm cartridges,flow rate 30 ml/min, acetonitrile/0.1% TFA gradient. The fractionscontaining the peptide are combined, concentrated in a rotary evaporatorand then lyophilized.

The peptide (0.025 mM) is dissolved in a 10 mM ammonium acetatesolution. The pH is adjusted to 8.5 with 1 M ammonium hydroxidesolution. The pH is readjusted after 3 or 4 hours. The cyclization ismonitored by HPLC at 214 nm and 280 nm, WATERS Delta Pak C18™ 5μ column,acetonitrile/0.1% TFA gradient. The cyclization is complete after 15hours. The pH is brought to 6 using 97-100% acetic acid, the solution islyophilized and then purified under the same conditions as for the crudepeptide.

The peptide is checked by HPLC and by mass spectrometry according to theelectrospray technique (FIGS. 18A-B and FIGS. 19A-B) (FISON VG Trio2000™ spectrophotometer).

Fmoc: 9-Fluoraenylmethyloxycarbonyl Pmc:8-Methylpentane-6-sulfonylchroman Trt: Tritryl Boc: TertbutyloxycarbonyltBU: tert butyl DMF: Dimethylformamide DIPCDI: DiisopropylcarbodiimideHOBT: 1-Hydroxybenzotriazole TFA: Trifluoroacetic acid Reagent K:Phenol/water/thioanisole/ethanedithiol/TFA; 2.5 ml/2.5 ml/ 2.5 ml/1.5ml/41 ml

It was found that some specific amino acids were conserved only betweengroup O viruses: such is the case for lysine in position 21 in a peptideof 26 amino acids, for threonine in position 7 and asparagine inposition 11. These differences could explain the absence of detection ofconventional HIV-1 envelope antigens by one of the sera from theHIV-1_((VAU)) patient and also probably by that of the patients infectedby other group O viruses. Overall, the comparison between the HIV-1-LAIand HIV-1_((VAU)) envelope sequences showed a 50% identity. TheHIV-1_((VAU)) envelope sequence was also compared to that of other HIVrepresentatives including the two members of HIV-1 group O described andsequenced: HIV-1_(ANT70) and HIV-1_(MVP5180) and SIV representatives.The results of this analysis, which are presented in Table 1, establishthat HIV-1_((VAU)) belongs to group O. The HIV-1_((VAU)) envelope is 70%identical to the HIV-1_(ANT70) envelope and 71% identical toHIV-1_(MVP5180). Among the most common HIV-1 subtypes, the identity atthe level of the envelope is comparable, ranging from 74% to 80%.

HIV-1_(EU) 77 HIV-1_(MAL) 76 80 HIV-1_(U455) 75 74 76 SIV_(CPZGAB) 61 6263 63 HIV-2_(ROD) 38 39 39 40 39 SIV_(MAC251) 37 37 38 38 38 74SIV_(AGMTYO) 37 36 37 38 40 46 47 HIV_(ANT70) 51 52 52 52 53 33 32 33HIV_(MVP5180) 51 52 54 51 52 36 33 34 70 HIV_(VAU) 50 51 52 51 54 35 3435 70 71 HIV-1_(LAI) HIV-1_(EU) HIV-1_(MAL) HIV-1_(U455) SIV_(CPZGAB)HIV-2_(ROD) SIV_(MAC251) SIV_(AGMTYO) HIV_(ANT70) HIV_(MVP5180)

The relationship between HIV-1_((VAU)), other members of the phylogenyof HIV-1 viruses and the two viruses of group O recently described wasanalyzed by constructing a phylogenetic tree of unweighted parsimonyusing the nucleotide sequence of the env transmembrane region. Theresult of this analysis is represented in FIG. 5 in which the numbersindicate the number of nucleotide changes. FIG. 5 shows thatHIV-1_((VAU)) is roughly equidistant from the other two group O virusesand that, overall, these three viruses appear to be approximatelyequidistant from each other. Indeed, the number of nucleotide changesbetween HIV-1_(MVP5180) and HIV-1_((VAU)) is 218 in the segment of thegenome analyzed, whereas the distance is 183 between HIV-1_(MVP5180) andHIV-1_(ANT70), and 213 between HIV-1_(ANT70) and HIV-1_((VAU)). Thisdivergence profile is very similar to that which exists in all the otherHIV-1 subtypes, where the number of single nucleotide changes whichexist between two different subtypes ranges from 157 (subtype E tosubtype F) to 219 (subtype A to subtype D).

Table 1 shows the comparison of the envelope sequences of differentviruses related to HIV-1. The numbers indicate the percentage of aminoacid identity between the envelope sequences, as calculated using theGAP program of the Wisconsin GCG package. *: in the case of HIV_(ANT70),only the external envelope protein was used in the comparison.

Compositions Comprising HIV-1_((VAU)) Antigens

Generally, the invention relates to any composition which can be usedfor the in vitro detection of the presence, in a biological fluid,especially from individuals who have been brought into contact withHIV-1_((VAU)), or with antibodies against at least one of theHIV-1_((VAU)) antigens. This composition can be applied to the selectivediagnosis of infection by an HIV-1 group O by using diagnostictechniques such as those described in Patent Applications EP 84401,834and EP 87400,151,4. Within the context of the present invention, anyconstituent comprising antigenic determinants capable of beingrecognized by antibodies produced against HIV-1_((VAU)) is used, forexample recombinant antigens or peptides or chemically synthesizedpeptides defined from the sequence of the HIV-1_((VAU)) envelope. Inthis regard, the invention relates more particularly to compositionscontaining at least one of the HIV-1_((VAU)) virus envelope proteins.There may be mentioned, by way of examples of compositions, those whichcontain proteins, glycoproteins or peptides from the envelope proteincorresponding to the entire 590-620 region of the HIV-1_((VAU)) gp41protein or to the parts of this region which are specific forHIV-1_((VAU)) such as the peptides -TFIQN- (SEQ ID NO:40) or -WGCKNR-(SEQ ID NO:41).

The invention also relates to compositions combining recombinant orsynthetic HIV-1_((VAU)) proteins and/or glycoproteins and/or peptideswith proteins and/or glycoproteins and/or peptides from HIV-1 and/orHIV-2 and/or from another HIV-1 group O which are obtained by extractionor in lysates or by recombination or by chemical synthesis and/orpeptides which are derived from these proteins or glycoproteins andwhich are capable of being recognized by antibodies induced by the HIV-1and/or HIV-2 and/or HIV-1 group O virus.

The diagnostic compositions containing antigenic determinants capable ofbeing recognized by antibodies directed against HIV-1_((VAU)), inparticular the peptide compositions, may be included in or combined withcompositions or kits already available for detecting infection by HIV-1and/or HIV-2 retroviruses, so as to extend the detection range of thekits to the detection of HIV-1 group O retroviruses.

By Way of Nonlimiting Examples:

-   -   either core proteins, particularly the gag, pol, HIV-1 and HIV-2        proteins or peptides thereof, and HIV-1_((VAU)), envelope        proteins or peptides,    -   or HIV-1 envelope glycoproteins, HIV-2 envelope glycoproteins        and HIV-_((VAU)), envelope glycoproteins,    -   or mixtures of HIV-1 proteins and/or glycoproteins, HIV-2        proteins and/or glycoproteins and HIV-1_((VAU)) envelope        proteins and/or glycoproteins.

It is important to note that, although the antibodies from patientsinfected with HIV-1 group O viruses react strongly with gag and polantigens from HIV-1 group M viruses, their reactivity is practicallyzero with group M virus envelope antigens. It is therefore importantthat the composition of the present invention comprise at least oneprotein or one peptide of the HIV-1_((VAU)) envelope so that this viruscan be detected with certainty.

Such compositions, when used in diagnosis, consequently help thediagnosis of AIDS or of the symptoms associated with it, which extendover a broader spectrum of causative etiological agents. It goes withoutsaying that the use of diagnostic compositions which contain onlyHIV-1_((VAU)) envelope proteins and/or glycoproteins is nonethelessuseful for the more selective detection of the category of retroviruswhich may be held responsible for the disease.

Methods and Kits for the Diagnosis of Infections Caused Especially bythe HIV-1_((VAU)) Virus

The present invention relates to a method for the in vitro diagnosis ofinfection caused by HIV viruses, etiological agents of AIDS and relatedsyndromes, which comprises bringing a serum or another biologicalmedium, obtained from a patient or subject being subjected to thediagnosis, into contact with a composition containing at least oneprotein, glycoprotein or peptide from HIV-1_((VAU)), and detecting apossible immunological reaction. Examples of such compositions weredescribed above.

Preferred methods involve, for example, immunofluorescence or ELISA typeimmunoenzymic reactions. The detections may be effected by direct orindirect immunofluorescence measurements or direct or indirectimmunoenzymic assays.

Such detections comprise for example:

-   -   depositing defined quantities of the extract or of the desired        antigenic compositions in accordance with the present invention        into the wells of a microplate;    -   introducing into each well a serum, diluted or undiluted, which        is capable of containing the antibodies, the presence of which        has to be detected in vitro;    -   incubating the microplate;    -   carefully washing the microplate with an appropriate buffer;    -   introducing into the wells of the microplate specific labeled        antibodies to human immunoglobulin, the labeling being performed        with an enzyme chosen from those which are capable of        hydrolyzing a substrate such that the latter then undergoes        modification of its absorption of radiation, at least in a        defined wavelength band, and    -   detecting, preferably in a comparative manner relative to a        control, the extent of the hydrolysis of the substrate as a        measure of the potential risks or of the actual presence of the        infection.

The present invention also relates to kits or boxes for the diagnosis ofHIV-1_((VAU)) virus infection, which comprise in particular:

-   -   an extract, a more purified fraction, or a synthetic antigen        derived from the types of viruses indicated above, this extract        fraction or antigen being labeled, for example, radioactively,        enzymically, fluorescently or otherwise,    -   antibodies to human immunoglobulins or a protein A (which is        advantageously attached to a support which is insoluble in        water) such as agarose beads for example) or microplate wells,        and the like),    -   optionally, a sample of biological fluid or cells obtained from        a negative control subject;    -   buffers and, where appropriate, substrates for visualizing the        label.

The subject of the invention is also immunogenic compositions which arecapable of inducing the formation of antibodies recognizing antigenswhich can be obtained by chemical synthesis or by recombination.

Serology

The capacity of the serum antibodies from the patient infected withHIV-1_((VAU)) to react with HIV-1 antigenic preparations was evaluatedusing various commercially available kits: Sanofi Diagnostics Pasteur,(Genelavia Mixt™) Abbott, Wellcome, and Behring. The reactivity of theseantibodies with various HIV-1 proteins was examined using the SanofiDiagnostics Pasteur Western-blot kit, following the proceduresrecommended by the manufacturer.

More precisely, the patient's serum was examined several times usingHIV-1 specific ELISA kits. It was first tested and proved to be positivein 1990, being noted 7.33 (this figure corresponds to the ratio of themeasured OD to the background OD) with the Sanofi. Diagnostics Pasteurkit, 3.50 with the Abbott kit and 2.70 with the Wellcome kit. During theuse of reagents specific both for HIV-1 and HIV-2, the serum was noted1.42 with the Behring kit and 4.40 with the Wellcome kit.

The capacity of the patient's serum to react on different dates withdifferent HIV-1 structural proteins was studied using the HIV-1LAV.BLOT™ immunoblot assay, a test marketed by Sanofi DiagnosticsPasteur. As shown in FIG. 5 with all the serum samples tested, only avery weak reactivity of the serum with the HIV-1 env proteins gp160 andgp120 was noted. However, the serum reacted strongly with the HIV-1 gagproteins p55 (gag precursor) and p24 (CA), and with the pol products p66(RT) and p34 (IN). By HIV-2 immunoblotting, only a very weak reactivitywas detected with the gag p26.

This illustrates that the detection of antibodies specific for group Owith commercially available serum diagnostic kits should be carefullycontrolled. Although serum antibodies from patients infected with groupO viruses show strong cross-reactions with the group M gag and polantigens, they show few or no reactions with the group M envelopeantigens. Consequently, it is possible to assume that a significantproportion of these patients might not be detected using some kits basedon group M envelope antigenic reagents. Indeed, in a recent preliminarystudy of several sera from patients infected with group O, it was foundthat the capacity to detect antibodies specific for group O was verydifferent depending on the detection kit used (Loussert-Ajaka, I., Ly,T. D., Chaix, M. L., Ingrand, D., Saragosti, S., Courrouce, A. M.,Brun-Vézinet, F. and Simon, F. (1994). HIV-1/HIV-2 seronegativity inHIV-1 subtype O infected patients. Lancet. 343, 1393-1394.). Thisimplies that a careful and extensive study of the reactivity of a largenumber of group O sera with all the diagnostic kits available on themarket is necessary.

Compositions comprising polyclonal or monoclonal antibodies preparedfrom recombinant or synthetic antigens from the HIV-1_((VAU)) virus.

The invention relates to a serum capable of being produced in animals byinoculating them with HIV-1_((VAU)), particularly the antigenic epitopesof HIV-1_((VAU)) and more particularly the antigenic epitopes of theHIV-1_((VAU)) virus envelope protein. The invention relates moreparticularly to the polyclonal antibodies more specifically orientedagainst each of the antigens, especially proteins or glycoproteins ofthe virus. It also relates to monoclonal antibodies produced by varioustechniques, these monoclonal antibodies being respectively oriented morespecifically against the various HIV-1_((VAU)) proteins, particularlythe HIV-1_((VAU)) envelope proteins.

These polyclonal or monoclonal antibodies can be used in variousapplications. There may be mentioned essentially their use forneutralizing the corresponding proteins, or even for inhibiting theinfectivity of the whole virus. They may also be used for example todetect viral antigens in biological preparations or to carry outprocedures for purifying the corresponding proteins and/orglycoproteins, for example during their use in affinity chromatographycolumns.

By way of example, anti-envelope antibodies or anti-gag antibodies arereagents which can be used in diagnosis, in particular for the detectionof HIV-1 group O particles by antigen capture ELISA.

The invention relates to antibodies directed against one or moreHIV-1_((VAU)) viral antigens produced from amino acid sequences ofHIV-1_((VAU)). Techniques for obtaining antibodies from antigenicepitopes similar to the antigenic epitopes of the HIV-1_((VAU)) virus ofthe present invention have been described previously.

The technique for the preparation of antibodies which is described inthe publication by Ulmer et al., 1993, may be used by persons skilled inthe art to prepare the antibodies of the present invention, themodifications which make it possible to adapt this technique to theantigens of the present invention forming part of the knowledge ofpersons skilled in the art.

Study of the Immunoreactivity of the vau Peptide

The immunoreactivity of the vau peptide was confirmed, afterexperimental ELISA plates had been prepared, according to a procedureestablished for a screening test for anti-HIV antibodies. This test isbased on the detection of a solid phase prepared with the peptide whichmimics the immunodominant epitope of the envelope glycoprotein of theHIV-1 group (or subgroup) O virus, VAU isolate. The implementation ofthe test was modeled on the procedure proposed by the Genelavia® Mixtkit, using the reagents in that kit. The experimental data collated inthe two tables of FIGS. 20A-C and 21A-C show that:

a) the four sera taken from patients contaminated with the HIV-1 group(or subgroup) O virus are very reactive with the vau peptide;

b) the ten sera supposedly taken from patients contaminated with theHIV-1 (group or subgroup) O virus, among the 19 sera sent out by thePasteur Institute of Yacoundé, are also highly reactive with this samepeptide;

c) the sera (4 samples) taken from individuals contaminated with theHIV-1 subtype B virus (in the acute phase) are not reactive with the vaupeptide;

d) the sera taken from asymptomatic blood donors (48 samples tested) arenot reactive with the vau peptide; These experimental data, althoughlimited (in view of the paucity of HIV-1 group (or subgroup O)antibody-positive samples), bear witness to the sensitivity andspecificity of the peptide selected.

From the above text, it follows that the invention also relates to thedetection of the HIV-1_((VAU)) virus or of a variant by virtue of theuse of the antibodies described above in a method involving variousstages, these stages being specifically intended to reveal thecharacteristic properties of the HIV-1_((VAU)) virus.

The invention also relates to the detection of the HIV-1_((VAU)) virusby molecular hybridization.

Generally, this method for detecting the HIV-1_((VAU)) virus or avariant in serum samples or samples of other biological fluids ortissues, obtained from patients liable to be carriers of theHIV-1_((VAU)) virus, comprises the following stages:

-   -   the manufacture of at least one optionally labeled probe;    -   at least one hybridization stage performed under conditions        permitting hybridization by bringing the nucleic acid of the        suspect patient's sample into contact with said labeled probe        and optionally immobilizing the complex formed on an appropriate        solid support,    -   where appropriate, washing said solid support with a suitable        washing solution,    -   the detection of said complex and therefore of the presence or        otherwise of the HIV-1_((VAU)) virus by an appropriate detection        method known to those skilled in the art.

In another preferred embodiment of the method according to theinvention, the abovementioned hybridization is performed undernonstringent conditions and the membrane is washed under conditionsadapted to those for the hybridization.

By using serology or a gene amplification technique such as specificPolymerase Chain Reaction (PCR), the extent of the HIV-1 group Oepidemic was precisely evaluated. It was found that 5 to 10% of patientsinfected with HIV-1 in Cameroon are in fact infected with group Oviruses. However, apart from the virus isolate described here, thepropagation of the group O virus outside West Central Africa has notbeen documented. The patient from whom HIV-1_((VAU)) was isolated hasalways lived in France and has never traveled to Africa. Up until now,we have no precise proof relating to the origin of her infection, butthis case indicates that a degree of propagation of the group O virusesin Europe has already occurred.

The invention also relates to a process of detection and discrimination,in a biological sample, between antibodies characteristic of an HIV-1group (or subgroup) O retrovirus and antibodies characteristic of aretrovirus of the HIV-1-M type, characterized by placing this biologicalsample in contact with a peptide which does not react with theantibodies characteristic of a retrovirus of the HIV-1-M type, inparticular one chosen from the peptides (1), (2), (3), (4), (5 a), (5b),(9) and (10) described above.

Also, the invention relates to a process of detection anddiscrimination, in a biological sample, between antibodiescharacteristic of an HIV-1 group (or subgroup) O retrovirus andantibodies characteristic of a retrovirus of the HIV-1-M type,characterized by placing this biological sample in contact with thepeptide derived from one of the HIV-1 M viruses taken into considerationin FIGS. 8 and 9 and homologous to a peptide chosen from the peptides(1), (2), (3), (4), (5a), (5b), (9) and (10), the sequence of thishomologous peptide resulting from vertical alignments of its ownsuccessive amino acids which are themselves contained in the pertinentpeptide sequence relative to the corresponding HIV-1-M virus andrepresented in FIG. 8 or 9 with the successive amino acids of thesequence of the peptide chosen, as also follows from FIG. 8 or 9.

According to the present invention, the process of detection anddiscrimination between infection by an HIV-1 group (or subgroup) Oretrovirus and an HIV-1 subgroup M retrovirus is characterized byplacing serum, obtained from individuals subjected to an AIDS diagnostictest, in contact, in particular, with the peptide RILAVERY (SEQ IDNO:35). In addition, the process for detection of infection due eitherto an HIV-1 subgroup O or HIV-1 subgroup M retrovirus is characterizedby the use of mixtures of two categories of peptides, those of the firstcategory corresponding to the peptides (1), (2), (3), (4), (5a), (5b),(9) and (10).

Moreover, the process for discrimination between an infection due to anHIV-1-O DUR retrovirus and an infection due to another type of HIV-1-Oretrovirus, is characterized by placing the biological test sample incontact with any of the peptides (11) to (15) or of the peptides (17) to(20).

Alternatively, it is a process for discrimination between an infectionby an HIV-1 group (or subgroup) O retrovirus and an HIV-1 subgroup Mretrovirus, using a serine protease whose cleaving action is carried outon an SR dipeptide, and comprising the detection of a cleavage or of anon-cleavage of the V3 loop of gp 120 of the retrovirus, depending onwhether this retrovirus is an HIV-1 group (or subgroup) O retrovirus oran HIV-1 subgroup M retrovirus.

The invention also relates to a composition for detection anddiscrimination, in a biological sample, between an HIV-1 subgroup Mretrovirus and an HIV-1 group (or subgroup) O retrovirus, comprising amixture of two categories of peptides, the first being in particularthose identified (1), (2), (3), (4), (5a), (5b), (9) and (10).

Monoclonal antibodies specific for the sequences of each of the peptides(1) to (20) also fall within the scope of the present invention.

The invention also relates to a plasmid chosen from those which weredeposited at the CNCM on 24 Feb. 1995 under the references I-1548,I-1549 and I-1550.

The invention is also directed towards nucleic acids containing asequence which codes for each of the peptides (1) to (20) defined in thepresent invention.

Among the preferred nucleic acid sequences, the nucleotide sequencesrepresented in FIG. 10, 11 or 12 will be chosen.

The invention also relates to vectors containing a nucleic acid asdefined above.

The invention is also directed towards cells liable to contain any oneof said nucleic acids or of said vectors.

The present invention also relates to a virus such as that deposited on23 Feb. 1995 at the CNCM under the reference I-1542.

A virus also falling within the scope of the invention is a virus of thesame group as the above, characterized in that consensus peptides ofthis virus are recognized by antibodies which specifically recognize apolypeptide or a peptide defined above.

The genomic RNA of this virus also falls within the scope of theinvention.

Also falling within the scope of the invention is a box or kit fordetection of antibodies in the serum or any other biological sample froma patient liable to be infected with a human retrovirus of the HIV type,characterized in that it comprises:

-   -   at least one polypeptide or one peptide having, in particular,        as its sequence one of the sequences (1) to (20) described        above.    -   means allowing the reaction for formation of the immunological        complex between the polypeptide(s) or the peptide(s) and the        antibodies which may be present in the biological sample to be        tested, for example one or more incubation buffers, if needed,    -   a negative control sample,    -   means for visualizing the antigen/antibody complex formed.

Also according to the invention, this kit contains, in addition, atleast one consensus peptide or polypeptide derived from another HIVstrain or from a peptide or polypeptide comprising

either an amino acid sequence which is separate from the sequence ofthis polypeptide or peptide, in which one or more amino acids arereplaced with other amino acids, with the proviso that the peptide orpolypeptide retains its reactivity with an antiserum against theconsensus peptide or polypeptide,

or an amino acid sequence in which one or more amino acids have beendeleted or added, with the proviso that the peptide or polypeptideretains its reactivity with an antiserum against the consensus peptideor polypeptide.

Preferably, a kit according to the invention will contain, in addition,at least one peptide or polypeptide derived from another HIV strain,preferably the HIV-LAI strain.

The invention also relates to a polypeptide composition for the in vitrodiagnosis of an infection due to a retrovirus according to theinvention, or to one of its variants, this diagnosis being made on abiological sample liable to contain antibodies formed after saidinfection. This composition is characterized in that it comprises atleast one of the peptides (1) to (20).

The biological sample may consist in particular of blood, plasma, serumor any other biological extract. The above compositions may be used forthe detection of antibodies in one of the abovementioned biologicalsamples.

The invention is therefore also directed toward a method for the invitro diagnosis of an infection due specifically to a retrovirus of theHIV type, characterized by the steps of:

-   -   placing a biological sample, which is liable to contain        antibodies produced after an infection by an HIV-1 group (or        subgroup) O retrovirus, in contact with a peptide as defined        above, or with a peptide composition as defined above, under        suitable conditions which allow the formation of an        immunological complex of the antigen/antibody type,    -   detection of the possible presence of the complex.

The invention moreover relates to an immunogenic composition,characterized in that it comprises at least one peptide in combinationwith a pharmaceutical vehicle which is acceptable for making upvaccines.

The invention also relates to a process for the preparation of capsidproteins and gp41 and gp 120 glycoproteins of a retroviral strainaccording to the invention, the process being characterized by thefollowing steps:

-   -   lysis of the cells infected with an HIV-1 retrovirus according        to the invention and separation of the supernatant and of the        infected cells or lysis of the viral pellets prepared by        centrifugation,    -   deposition of the cell extract and/or of the viral extract on an        immunoadsorbant containing purified antibodies, which are        obtained from the serum of an individual infected by a        retrovirus according to the invention and advantageously        attached to a suitable support, said serum of the infected        individual having the capacity to react strongly with envelope        proteins of the virus according to the invention,    -   incubation in the presence of a buffer and for a sufficiently        long period to obtain the formation of an antigen/antibody        immunological complex,    -   washing of the immunoadsorbant with a buffer in order to remove        the molecules not retained on the support,    -   recovery of the desired antigenic proteins. According to a first        embodiment of this preparation process, the separation and the        recovery of the capsid proteins and of the gp41 and gp120        glycoproteins of HIV-1 DUR are carried out by electrophoresis        and by electroreduction of the proteins.

According to another embodiment of this preparation process, theproteins are recovered by:

-   -   elution of the proteins attached to the above immunoadsorbant,    -   purification of the products thus eluted on a chromatography        column containing, attached to the separation support,        antibodies which recognize the capsid proteins and the gp41 and        gp120 glycoproteins of HIV-1 group (or subgroup) O DUR.

Also falling within the scope of the invention is a process for theproduction of a peptide or polypeptide according to the invention, thispeptide or polypeptide being obtained

-   -   either by expression of a nucleic acid of the invention,    -   or by chemical synthesis, by addition of amino acids until this        peptide or this polypeptide is obtained.

The standard principles and processes of genetic engineering may be usedhere (“Molecular cloning”, Sambrook, Fritsch, Maniatis, CSH 1989).

Also falling within the scope of the invention is a process for theproduction of a nucleic acid defined above, which may be produced eitherby isolation from a virus of the invention, or by chemical synthesis, orby using techniques of in vitro amplification of nucleic acids fromspecific primers.

Oligonucleotide primers also according to the invention have a sequenceconsisting of at least eight consecutive nucleotides of the followingnucleotide sequences:

ATT CCA ATA CAC TAT TGT GCT CCA-3′ (SEQ ID NO: 42) AAA GAA TTC TCC ATGACT GTT AAA-3′ (SEQ ID NO: 43) GGT ATA GTG CAA CAG CAG GAC AAC-3′ (SEQID NO: 44) AGA GGC CCA TTC ATC TAA CTC-3′. (SEQ ID NO: 45)

These primers may be used in a gene amplification process, for exampleby PCR or an equivalent technique, of a nucleotide sequence coding for apeptide of the invention. Tests carried out with these primers gaveconclusive results.

Also, the invention relates to a kit allowing amplification by PCR or anequivalent technique described above.

Also falling within the scope of the present invention is a process fordetection of the presence, in a biological sample, of nucleic acid(s)characteristic of an HIV-1 group (or subgroup) O DUR retrovirus,including a retrovirus according to the invention. This processcomprises a placing in contact of a cDNA formed from RNA(s) contained inthis biological sample, under conditions allowing the hybridization ofthis cDNA with the retroviral genome, and the execution of a geneamplification on this viral sample:

The invention also relates to a viral lysate as obtained by lysis ofcells infected with a virus according to the invention.

A protein extract from an HIV-1_((DUR)) (or HIV-1_((VAU))) straincontaining in particular a peptide or a polypeptide as defined abovealso falls within the scope of the-invention.

The invention relates to specific peptides obtained from the structureof HIV-1 group (or subgroup) O DUR or from variants of this retrovirusand which make it possible either to discriminate, depending on thecase,

-   -   globally between HIVs-1 belonging to the category O and HIVs-1        belonging to the category M,    -   or, more specifically, between viruses belonging to the subgroup        characteristic of the HIV-1 group (or subgroup) O DUR and other        viruses of the subgroup O,

or, on the other hand, to recognize most, if not all, of theretroviruses both of the group (or subgroup) O and of the subgroup M.

Also falling within the scope of the invention are the correspondingpeptides obtained from corresponding structural proteins of other HIV-1group (or subgroup) O or HIV-1 subgroup M viruses, in particular thosederived from the GAG, gp120 and gp41 structural proteins whose parts areindicated in the diagrams, these homologous peptides ensuing from theirbeing placed in alignment, as also results from the diagrams with thepeptides obtained from the HIV-1 group (or subgroup) O DUR, moreparticularly identified in the present text. Similarly, certainhomologous peptides may be used in those tests which make it possible tocarry out the abovementioned discriminations, it being understood inthis case that they are then used in place of the correspondingpeptides, derived from the GAG, gp120 and gp41 structural proteins.

Determination of Oligonucleotides Specific for the O Group

Using the VAU sequence and its correlation with the MVP5180 and ANT70sequences, oligonucleotide primers were defined which endeavor to bespecific for the subgroup in its entirety for the V3 region and the gp41region. These primers made it possible to amplify the DUR strain andconsequently constituted one solution to the amplification problemencountered. The position and the sequence of these HIV subgroup Oprimers are represented in FIGS. 13B and A, respectively. These primersmake it possible to obtain an amplification band which is visible onstaining with ethidium bromide, with a single step of 30 cycles of PCR.Partial sequences were obtained:

-   -   GAG: 513 base pairs (171 amino acids)=SEQ ID NO:95 and SEQ ID        NO:96, FIGS. 10A and B    -   gp120 V3 loop: 525 base pairs (75 amino acids)=SEQ ID NO:97 and        SEQ ID NO:98, FIGS. 11A and B    -   gp41 immunodominant region: 312 base pairs (104 amino acids)=SEQ        ID NO:99 and SEQ ID NO:100, FIGS. 12A and B.

Nucleotide (FIGS. 15A-C) and protein (FIGS. 16A-C) comparisons of theDUR sequences with the MVP5180, ANT and VAU sequences for the Osubgroup, LAI for the HIV-1 consensus sequence, representative AfricanHIV-1 MAL sequence, and CPZ for the CIV of the Gabonese chimpanzee, showthat DUR is as remote from the otter published HIV-1 group (or subgroup)O strains as the latter are from each other.

The differences are less in the GAG region and maximal in the region ofthe V3 loop of gp120, where the protein comparison reaches a differenceof 400 (FIG. 16). The phylogenic trees confirm, on the one hand, thatthe DUR strain forms part of the O subgroup, and, on the other hand, theimportance of the differences between the various O strains described,without, however, subtype branching emerging clearly (FIG. 17).

Comparison of the GAG Sequences:

On comparison of the GAG sequence obtained with the other two O strainspublished, ANT70 and MVP5180, as well as with the representativesequences of the M group (FIG. 8), it was possible to observe that an Oconsensus sequence exists in several regions, which is distinct from theM consensus sequence in the same regions. Two hypervariable regions,which are more variable for O than for M, and a few point differencesfor one or the other strain may also be found. Nevertheless, the regionsSPRT (SEQ ID NO: 104) . . . SEGA (SEQ ID NO: 105), MLNAI (SEQ ID NO:106) . . . KEVIN (SEQ ID NO: 107), GPLPP (SEQ ID NO: 108) . . . QQEQI(SEQ ID NO: 109) and VGD . . . SPV appear to discriminate between the Oconsensus sequence and the M consensus sequence.

The regions QQA and LWTTRAGNP (SEQ ID NO:9) are hypervariable regions.The HIV-1 group (or subgroup) O DUR strain is conspicuously different inthree positions with respect to the M and O consensus sequence (L for Iand twice for E) and takes a specific amino acid in three isolatedhypervariable positions, V position L9; A position A77; L position 110.

In addition, it is possible to define in the GAG region segments commonto the O group and to the M group, such as SPRTLNAWVK (SEQ ID NO: 15),GSDIAGTTST (SEQ ID NO:16) and QGPKEPFRDYVDRF (SEQ ID NO:17).

Comparison of the Sequences of the V3 Loop

This comparative study revealed considerable differences of up to 56%for protein differences with the HIV-1 subgroup M consensus sequence,and 35 to 42% with the other HIV-1 group (or subgroup) O consensussequences.

The alignments of peptide sequences in the regions of the V3 loop of gp120 and in the immunodominant region of gp41 is given in FIG. 9. Thesequence of the interior of the V3 loop of the DUR strain differssubstantially from that of the HIV-1 subgroup M consensus sequence. Itshares the motif GPMAWYSM (SEQ ID NO:28) with the VAU and ANT70 strainsbut not with the MVP strain, which has two substitutions: R for A and Rfor Y.

The left and right parts of the rest of the V3 loop are markedlydifferent from all the other HIVs known and do not leave it possible toimagine other cross-reactivities. Furthermore, the V3 loop of DUR is oneamino acid longer than the other O sequences, which are themselvesanother one amino acid longer than the sequences of the HIV-1 M group.

Comparison of the Alignments Concerning the Immunodominant Region ofgp41

The “mini loop” of the DUR strain, having the sequence CRGKAIC (SEQ IDNO: 110), proved to be very specific for this strain: it mightconstitute an epitope (see FIG. 9). In addition, this sequence might belikely to be involved in the modification of the conditions of unfoldingof the gp41 glycoprotein, and consequently in the infectiousness of thestrain.

A long sequence of 11 amino acids flanking this loop on the left isidentical to the VAU sequence. A polymorphism of the DUR strain may benoted for an S or T position according to the clones analyzed.

Corresponding peptides obtained from other known retroviral strains arealso represented in FIG. 9.

The DUR strain also makes it possible to define an HIV subgroup Oconsensus sequence of the gp41 region, several sufficiently longhomologous regions of which might be used. These homologous regions are,inter alia: RL*ALET (residues 1-7 of SEQ ID NO: 92), QNQQ (residues10-13 of SEQ ID NO: 92), LWGC (residues 17-20 of SEQ ID NO: 92) andCYTSV (residues 26-30 of SEQ ID NO: 92) (*representing a variable aminoacid).

Serological Correlations:

The anti-DUR antiserum does not react with the peptides of the V3 loopof the HIV-1-M consensus sequence, of HIV-1 MAL, of HIV-1 CPZ or ofHIV-1 group (or subgroup) O MVP5180 but does, however, react with thepeptide of the V3 loop of HIV-1-O ANT70 as seen in FIG. 14A. As regardsthe gp41 immunodominant region, this does not react with the “standard”HIV-1 subgroup M consensus sequence as seen in FIG. 14B, but does,however, react, weakly but surprisingly, with the HIV-1 subgroup Mright-extended consensus sequence.

References

Agut, H., Candotti, D., Rabanel, B., Huraux, J., Remy, G., Ingrand, D.,Tabary, T., Chippaux, C., Chamaret, S., Guetard, D., Dauguet, C. andMontagnier, L. (1992). Isolation of atypical HIV-1-related retrovirusfrom AIDS patient. Lancet. 340, 681-682.

Alizon, M., Wain-Hobson, S., Montagnier, L. and Sonigo, P. (1986).Genetic variability of the AIDS virus: nucleotide sequence analysis oftwo isolates from African patients. Cell. 46, 63-74.

Barré-Sinoussi, F., Chermann, J. C., Rey, F., Nugeyre, M. T., Chamaret,S., Gruest, J., Dauguet, C., Axler-Blin, C., Brun-Vezinet, F., Rouzioux,C., Rozenbaum, W. and Montagnier, L. (1983). Isolation of aT-lymphotropic retrovirus from a patient at risk for acquired immunedeficiency syndrome (AIDS). Science. 220, 868-871.

Benn, S., Rutledge, R., Folks, T. et al, e. (198-5). Genomicheterogeneity from AIDS retroviral isolates from North America andZaire. Science. 230, 949-951.

-   Clavel, F. and Chameau, P. (1994). Fusion from without directed by    Human immunodeficiency virus particles. J. Virol. 68, 1179-1185.

Clavel, F., Guétard, D., Brun-Vézinet, F., Chamaret, S., Rey, M. A.,Santos-Ferreira, M. O., Laurent, A. G., Dauguet, C., Katlama, C.,Rouzioux, C., Klatzmann, D., Champalimaud, J. L. and Montagnier, L.(1986). Isolation of a new human retrovirus from West African patientswith AIDS. Science. 233, 343-346.

De Cock, K. M., Adjorlolo, G., Epkini, E., Sibailly, T., Kouadio, J.,Maran, M., Brattegaard, K., Vetter, K., Doorly, R. and Gayle, H. (1993).Epidemiology and transmission of HIV-2: why there is no HIV-2 epidemic.JAMA. 270, 2083-2086.

De Leys, R., Vanderborght, B., Vanden Haesevelde, M., Heyndrïckx, L.,van Geel, A., Wauters, C., Bemaerts, R., Saman, E., Nijs, P. andWillems, B. (1990). Isolation and partial characterization of an unusualhuman immunodeficiency retrovirus from two persons of west-centralAfrican origin. J. Virol. 64, 1207-1216.

Gnann, J., Cormick, J., Michell, S., Nelson, J. and Oldstone, M. (1987).Synthetic peptide immunoassay distinguishes HIV type 1 and type 2infections. Science 237, 1346-1349.

Grez, M., Dietrich, U., Balfe, P., von Briesen, H., Maniar, J.,Mahambre, G., Delwart, E., Mullins, J. and Rubsamen-Waigmann, H. (1994).Genetic analysis of Human Immunodeficiency virus type 1 and 2 (HIV-1 andHIV-2) mixed infections in India reveals a recent spread of HIV-1 andHIV-2 from a single ancestor for each of these viruses. J. Virol. 68,2161-2168.

Gürtler, L. G., Hauser, P. H., Eberle, J., von Brunn, A., Knapp, S.,Zekeng, L., Tsague, J. M. and Kaptue, L. (1994). A new subtype of HumanImmunodeficiency Virus type 1 (MVP-5180) from Cameroon. J. Virol. 68,1581-1585.

Harada, S., Koyanagi, Y. and Yamamoto, N. (1985). Infection ofHTLV-III/LAV in HTLV-I-carrying cells MT-2 and MT-4 and application in aplaque assay. Science. 229, 563-566.

Hirt, B. (1967). Selective extraction of polyoma DNA from infected mousecell cultures. J. Mol. Biol. 26, 365-369.

Huct, T., Cheynier, R., Meyerhans, A., Roclants, G. and Wain-Hobson, S.(1990). Genetic organization of a chimpanzee lentivirus related toHIV-1. 345, 356-359.

Javaherian, K., Langlois, A. J., LaRosa, G. J., Profy, A. T., Bolognesi,D. P., Herlihy, W. C., Putney, S. D. and Matthews, T. J. (1990). Broadlyneutralizing antibodies elicited by the hypervariable neutralizingdeterminant of HIV-1. Science. 250, 1590-1593.

Javaherian, K., Langlois, A. J., Mc Danal, C., Ross, K. L., Eckler, L.I., Jellis, C. L., Profy, A. T., Rusche, J. R., Bolognesi, D. P.,Putney, S. D. and Matthews, T. J. (1989). Principal neutralizing domainof the human immunodeficiency virus type 1 envelope protein. Proc. Natl.Acad. Sci. USA. 86, 6768-6772.

Louwagie, J., McCutchan, F., Peeters, M., Brennan, T., Sanders-Buell,E., Eddy, G., van der Groen, G., Fransen, K., Gershy-Damet, G. andDeleys, R. (1993). Phylogenetic analysis of gag genes from 70international HIV-1 isolates provides evidence for multiple genotypes.AIDS. 7, 769-80.

Louwagie, J., McCutchan, F., Van der Groen, G., Peeters, M., Fransen,K., Piot, P., Gershy-Damet, G., Roelants, G., Van Heuverswyn, II. andEddy, G. (1992). Genetic comparison of HIV-1 isolates from Africa,Europe, and North America. AIDS Res Hum Retroviruses. 8, 1467-9.

Matsushita, S. M., Robert-Guroff, M., Rusche, J., Koito, A., Hattori,T., Hoshino, H., Javaherian, K., Takatsuki, K. and Putney, S. (1988).Characterization of a Human immunodeficiency virus neutralizingmonoclonal antibody and mapping of the neutralizing epitope. J Virol 62,2107-2114.

NKENGASONG, J. N. et al., AIDS 1993, Vol. 7, No. 11, pp. 1536-1538.

Rey, M. A., Krust, B., Laurent, A. G., Montagnier, L. and Hovanessian,A. G. (1989). Characterization of human immunodeficiency virus type 2envelope glycoproteins: dimerization of the glycoprotein precursorduring processing. J. Virol. 63, 647-658.

ULMER J. B. et al., Science Vol. 259, March 1993, pp. 1745-1749.

Vanden Heasevelde, M., Decourt, J. L., de Leys, R. J., Vanderborght, B.,van der Groen, G., van Heuverswijn, H. and Saman, E. (1994). Genomiccloning and complete sequence analysis of a highly divergent AfricanHuman Immunodeficiency Virus isolate. J. Virol. 68, 1586-1596.

Vartanian, J.-P., Meyerhans, A., Asjö, B. and Wain-Hobson, S. (1991).Selection, recombination, and G→A hypermutation of HIV-1 genomes. J.Virol. 65, 1779-1788.

Wain-Hobson, S., Sonigo, P., Danos, O., Cole, S. and Alizon, M. (1985).Nucleotide sequence of the AIDS virus, LAV. Cell 40, 9-17.

1. An isolated or purified peptide consisting of the sequenceRL*ALET**QNQQ*L*LWGC*****CYTSV*WN*TW* (SEQ ID NO: 92), or a fragment ofthis sequence comprising at least two amino acid sequences selectedfrom: RL*ALET (residues 1-7 of SEQ ID NO: 92); QNQQ (residues 10-13 ofSEQ ID NO: 92); LWGC (residues 17-20 of SEQ ID NO: 92); CYTSV (residues26-30 of SEQ ID NO: 92); and WN*TW (residues 32-36 of SEQ ID No: 92);wherein * denotes any amino acid.
 2. The peptide of claim 1, whichcomprises the amino acid sequences RL*ALET (residues 1-7 of SEQ ID NO:92) and QNQQ (residues 10-13 of SEQ ID NO: 92).
 3. The peptide of claim1, which comprises the amino acid sequences RL*ALET (residues 1-7 of SEQID NO: 92); QNQQ (residues 10-13 of SEQ ID NO: 92); and LWGC (residues17-20 of SEQ ID NO: 92).
 4. The peptide of claim 1, which comprises theamino acid sequences RL*ALET (residues 1-7 of SEQ ID NO: 92); QNQQ(residues 10-13 of SEQ ID NO: 92); LWGC (residues 17-20 of SEQ ID NO:92); and CYTSV (residues 26-30 of SEQ ID NO: 92).
 5. An isolated orpurified peptide consisting of the amino sequenceRL*ALET**QNQQ*L*LWGC*****CYTSV*WN*TW* (SEQ ID NO: 92), wherein * denotesany amino acid.
 6. An isolated or purified peptide consisting of theamino acid sequence RLLALETLMQNQQLLNLWGCRGKAICYTSVQWNETWG (SEQ ID NO:90).
 7. A composition comprising a peptide as claimed in claim 1 and apharmaceutically acceptable carrier therefor.
 8. The peptide of claim 1,which comprises the amino acid sequences LWGC (residues 17-20 of SEQ IDNO: 92), CYTSV (residues 26-30 of SEQ ID NO: 92), and WN*TW (residues32-36 of SEQ ID NO: 92).
 9. The peptide of claim 1, which comprises theamino acid sequences LWGC (residues 17-20 of SEQ ID NO: 92) and CYTSV(residues 26-30 of SEQ ID NO: 92).
 10. An isolated or purified peptidewhich comprises a fragment of SEQ ID NO:92 and which has the followingsequence: WGC*****CYTSVQWN*T.
 11. An isolated peptide containing a partof the peptide LNLWGCRGKAICYTSVQWNETWG (18) (SEQ ID NO: 30) andcontaining the sequence SVQWN (20) (SEQ ID NO: 32).